Freezing Worm Strains
There is a good, complete protocol for freezing worm strains here. I've pasted it into the wiki here as well- credit: Morimoto Lab, Northwestern University. Freezing'' C. elegans'' Strains Reagents Needed: M9 (common stock)and Worm Freezing Solution Procedure: Make sure you begin with healthy, non-contaminated, non-starved worms of the correct phenotype. 1. Pick 20 young adult worms onto each of two (or three) 10 cm plates that have been seeded with 1 mL OP50 (40 or 60 worms total). If you are growing the worms at 15ºC, they will be ready to freeze in 1week. If you are growing the worms at 20ºC, they will be ready in about 4 days. 2. When the plates are ready to freeze, they should have: a. Little or no food (just starved) b. Plenty of L1s and L2s (these are what will survive) c. Eggs on the plate (means that the plate has not been without food for long) d. Worms with the correct phenotype e. No contamination (if contaminated, the worms will continue to eat and grow so there will be no stalling at the L1 stage) 3.Add about 5 mL of M9 to each plate. 4. Give each plate a swirl to loosen worms still stuck to the agar and then tilt plates so the liquid drains to one side of the plate. 5. Using a steripipette, collect the liquid (worms and M9) in a 15 mL conical tube (about 10 mL total). 6. Pellet the worms for about 1 minute at 1000 rpm in our Tabletop Centrifuge. 7. Aspirate as much of the supernatant as possible without disturbing the pellet. 8. Add about 15mL of M9 and resuspend the pellet. 9. Again, spin for about 1 minute at 1000 rpm. Repeat steps 7 and 8 if desired. 10. Aspirate all but about 3 mL (or 4.5 mL when using three plates) of M9. 11. Add an equal amount of Worm Freezing Solution 12. Briefly agitate the vial to suspend the worms and aliquot the worm suspension into 6 cryovials (9 cryovials with three plates) clearly labeled with the strain name, your initials and the date. 13. The styrofoam container that 15 mL conical tubes comes in is useful for freezing worms. The insulation provides the slow decrease in temperature required for survival. Place the cryovials into the styrofoam, and cover with another. Secure with tape or rubber bands and label the outside for future reference. 14. Store in your –80°C freezer space. 15. Test thaw the worms about 1 month later to ensure a successful freeze. A good freeze is when at least more than 10 worms survive. Pick several survivors on to a fresh plate to make sure they can produce progeny of the correct phenotype. Be sure to maintain the line while waiting for the results of the test thaw. SOLUTIONS NEEDED: M9 (1L) 5.8g Na2HPO4*7H2O P/N: (Sigma-Aldrich, S9390-1KG) 3.0g KH2PO4 P/N: (VWR, BDH0268-5006) 5.0g NaCl P/N: (EMD, SX0420-3) 0.25g MgSO4*7H2O P/N: (Bioworld, 413000-40-1) Milli-Q H2O to 1L Millipore filter P/N: (company, item #) (<0.22um) and bottle. Worm Freezing Solution (1L) 5.8g NaCl P/N: (EMD, SX0420-3) 50ml 1M KH2PO4 (pH 6.0) P/N: (VWR, BDH0268-5006) 240ml glycerol P/N: (Sigma-Aldrich, G7893-2L) 710ml milli-Q H2O -bottle and autoclave -after autoclaving add 30ul of sterile 1M MgSO4 (made using MgSO4*7H2O P/N: (Bioworld, 413000-40-1)) per 100ml of solution Cryovials (1.5ml Screw-Cap Tubes) P/N: SARSTEDT 72.692.100